This continuing research is aimed at characterizing, with micro- and nanoscale resolution, the collective molecular events of cellular signaling that are initiated by plasma membrane receptors and are highly orchestrated in space and time. Focus remains primarily on the high affinity receptor for IgE, Fc?RI, on mast cells, which plays a pivotal role in the allergic immune response and serves as a valuable model system for receptor-mediated cellular signaling. The primary goal of these studies is to elucidate structural interactions of proteins and lipids occurring within the plasma membrane milieu that are altered by antigen crosslinking of IgE- Fc?RI and result in transmembrane triggering of intracellular signaling cascades, culminating in cellular responses to the stimulant. Proposed research will investigate mechanisms by which the actin cytoskeleton regulates these interactions at multiple levels, including membrane pinning and compartmentalization by the cortical meshwork that prevents spontaneous initiation of signaling and rearrangements associated with cell adherence to a substrate that adjust the level of response. Specific Aim 1 will use two-color super resolution fluorescence microscopy to image and analyze dynamic interactions among signaling components in the plasma membrane that are initiated immediately after IgE-Fc?RI crosslinking, and regulation of these will be evaluated by selective perturbation to membrane and cytoskeleton. Structurally defined ligands will be employed to discern features of crosslinking that are critical for signal initiation, and these experimental results will be incorporated into computational models of signaling systems. Specific Aim 2 will use versatile micro- patterned surfaces and fluorescence microscopy to investigate later-stage membrane interactions initiated by clustered IgE-Fc?RI leading to downstream signaling and cellular response events that are modulated by integrin engagement. Selective inhibitors and molecular tension sensors will be employed to probe participation of the actin cytoskeleton and integrins in imposing mechanical forces and regulating signaling networks, as related to consequent cellular responses in single cell assays. Specific Aim 3 will translate biophysical insight and high-resolution approaches gained from detailed characterization of the IgE-Fc?RI model system to investigate plasma membrane interactions that participate in neurological cell activities and are disrupted in neurodegenerative diseases. The goal of these highly collaborative studies is to lay the groundwork and establish fruitful directions for significant biomedical impact. Initial experiments will evaluate cellular interactions of ?-synuclein that may occur in Parkinson's disease, cell surface clustering of neurotrophin receptor p75 related to signaling in neuronal plasticity, and lysosomal synapses that appear to be formed by microglia to hydrolyze A? fibrils associated with Alzheimer's disease.